Dylight 594 phalloidin

VSMCs were seeded into a 24well plate at a density of 3 × 10⁴ cells/ml and cultured for 24 h. Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 10 min at RT. After washing, cells were blocked with 2% BSA in PBS for 1 h, removed from the blocking solution, and incubated overnight at 4 °C with rabbit anti-Nrf2 (1:50) (Cell Signaling, Beverly, MA, US), rabbit anti-AhR (1:100) (BioWord, MN, US), rabbit-anti-FAK (1:100) (Cell Signaling, Beverly, MA, US), mouse anti-paxillin (1:300) (Sigma, MO, US), mouse anti-integrin β1 (1:100) (Sigma, MO, US) and actin stains with DyLight™ 594 Phalloidin (1:20) (Cell Signaling, Beverly, MA, US). Then, the cells were gently washed under the cover slip three times with PBS and mounted in fluoroshield™ mounting medium with DAPI (Cell Signaling, Beverly, MA, US). Cells were visualized under a laser scanning confocal microscope (Fluoview FV1000 confocal system, Olympus). To ensure blinded data analysis, immunofluorescence staining were performed by one person, and all images and samples were recorded by another person.

Cells were fixed in 4% formaldehyde, permeabilized with 0.5% Triton X-100, and probed with the specific primary antibodies against Smad3 (Cell Signaling Technology), N-cadherin (Cell Signaling Technology), and E-cadherin (Cell Signaling Technology) overnight at 4°C, followed by incubation with secondary antibody conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen). The nuclei were counterstained with DAPI. Actin filaments were stained with DyLight 594 phalloidin (Cell Signaling Technology). Images were obtained using confocal microscopy (Leica Microsystems, Germany). To quantitatively evaluate the nuclear translocation of Smad3, cells with positive nuclear Smad3 staining were calculated in 10 random fields under a ×40 objective, and then the quantity for each sample was summed up. To analyze the co-localization of lnc-TSI and Smad3, 786-O cells were subjected to a FISH assay, then fixed for 15 min in 4% formaldehyde and subjected to immunofluorescence staining.

To visualize TRPV4 localization, cells were transiently transfected with a plasmid encoding TRPV4-YFP, using Fugene HD (E2311, Promega) per manufacturer's instructions. Cells were then washed with PBS and fixed for 10 min with 4% paraformaldehyde (PFA). Cells were permeabilized with 0.01% TritonX-100 and labeled with DyLight 594 Phalloidin, as per manufacturers instructions (12877, Cell Signaling Technology). Cells were imaged using either a Zeiss Axioskop or Leica SP8 with a 40x objective. Resulting images were analyzed using ImageJ analysis software (Schneider et al., 2012 (link)). In order to assess the localization of TRPV4 and actin to pillar structures, each pilus was scored according to whether the protein formed a ring around the total circumference of the pilus, a partial circumference or if there was no increase in signal around the pilus. The TRPV4-YFP and phalloidin-594 signals were then compared and scored as a colocalization when similar structures were observed around an individual pilus.

CLL cells (20 × 10⁶/ml) were seeded on poly-L-lysine–coated glass and then left either inactivated or activated with F(ab′)2 Fragment anti-IgM or F(ab′)2 Fragment anti-IgG at 10 µg/ml (Jackson ImmunoResearch, PA, USA) for 5, 15, and 40 min. The cells were fixed with 4% methanol-free paraformaldehyde for 20 min, followed by permeabilization with 0.2% TX-100 (Sigma Aldrich, MO, USA). The antibodies used were fluorescein isothiocyanate (FITC)-conjugated anti-IgM, FITC-conjugated anti-IgG (Bethyl, TX, USA) (1:200) for 60 min, and DyLight 594 Phalloidin (Cell Signaling Technology, MA, USA) (1:20) for 20 min. Phospho-CD79a (Tyr182) (1:200) and anti-rabbit IgG (H+L), F(ab′)2 Fragment (Alexa Fluor^® 594 Conjugate) (1:500), Cell Signaling Technology (Beverly, MA), were used for pCD79a staining. LAMP1 (1:100) and anti-mouse IgG (H+L), F(ab′)2 Fragment (Alexa Fluor^® 594 Conjugate) (1:500), were used for LAMP1 staining. After incubation, cells were washed three times with 1% bovine serum albumin (BSA) in PBS. All the cells were labeled with fluorescent mounting medium with DAPI (Golden Bridge International, Inc., WA, USA). Images were acquired using Leica SP8 confocal microscope (Sackler Faculty of Medicine core facility, Tel Aviv University). Quantification was performed using Imaris (version 9.3.1) software.